This proposal seeks to continue a successful line of investigation into how skin and microbial proteases modify antimicrobial peptide function. Previous data have shown that activation of cathelicidin antimicrobial peptides is dependent on serine proteases including Kallikrein 5 and 7. Alternative processing of the substrate cathelicidin precursor hCAP 18 results in a variety of mature peptides with alternative function. Control of this processing is relevant to skin diseases ie. rosacea and psoriasis. Additional processing by microbial proteases serves as a virulence mechanism to alter normal host immunity. Recent data have shown alternative enzymatic processing of cathelicidin enables keratinocytes to recognize self-DNA through TLR9 and that TLR9 is essential for defense against skin infection by Group A Streptococcus. These findings suggest a novel role for nucleic acid recognition by skin epithelium. The focus of our proposal is to define what proteolytic products of cathelicidin processing control pattern. This approach will also extend to evaluation of how microbes influence these events. Specific Aim 1. Define the structure-function relationships between cathelicidin peptides and the capacity of keratinocytes to respond to CpG and genomic DNA. We will generate a library of cathelicidin peptides and test their ability to influence TLR9 activation. This screen will define the critical structural elements of the peptide and identify key enzymatic steps that result in the generation of these products. Specific Aim 2. Characterize the immune response to products defined in SA1 to determine the consequences of this to skin inflammation. We will establish the physiological relevance of peptides identified in aim 1 by using mouse models of skin inflammation, examine how the products of keratinocytes stimulated by alternative cathelicidin peptides alter T cell polarization, and establish the role of these findings in the in vivo skin inflammatory response. Specific Aims3. Determine how protease and nuclease expression by a bacterial pathogen modulates host cutaneous innate immune responses. We will study how protease expression by a bacterial pathogen changes the size and activity profile of cathelicidin peptides in skin. We will investigate cathelicidin expression in DNA-based extracellular traps (ETs) produced by neutrophils and mast cells and their effect on keratinocyte TLR-9 signaling. We will study how bacterial protease and nuclease expression influences these interactions, and how cathelicidins themselves may promote ET production.